Pharmaceutical formulations comprising a clavulanic acid salt and erithromycin derivatite

ABSTRACT

A pharmaceutical formulation comprising a pharmaceutically acceptable salt of clavulanic acid in combination with erythromycin and another antimicrobial agent useful for the treatment of an infection by intracellular pathogens in humans or animals.

This invention relates to pharmaceutical formulations, in particular tonovel uses of formulations in connection with the treatment of infectionby intracellular pathogens, in particular microorganisms of the genusLegionella, particularly L. pneumophila.

Intracellular pathogens include microorganisms of the genus Legionella,Chlamydia (e.g. C. trachomatis and C. pneumoniae) and Mycobacterium (e.gM. avium, M. fortuitum and M. tuberculosis). Legionella spp. are knownto infect the respiratory tract of humans and animals, causing acute andsometimes fatal symptoms. It is known to treat Legionella infection withantibiotics, for example erythromycin. It is an object of this inventionto provide alternative and improved pharmaceutical treatments forLegionella infection.

The invention provides a pharmaceutical formulation which comprises apharmaceutically acceptable salt of clavulanic acid, in combination witherythromycin or a derivative thereof, and optionally one or more otherantimicrobial agents.

The formulation of the invention is suitable for use in the treatment ofinfection by intracellular pathogens, e.g. microorganisms of the genusLegionella, in humans or animals.

The present invention also provides a method of use of apharmaceutically acceptable salt of clavulanic acid and erythromycin ora derivative thereof, and optionally one or more other antimicrobialagents, together in combination in the manufacture of a medicamentformulation, particularly a formulation for the treatment of infectionof humans or animals by intracellular pathogens such as microorganismsof the genus Legionella.

The present invention further provides a method for the preparation of apharmaceutical formulation as defined above, which method comprisesadmixing the combination of a pharmaceutically acceptable salt ofclavulanic acid, and erythromycin or a derivative thereof and optionallyone or more other antimicrobial agents.

As salts of clavulanic acid are extremely hygroscopic such formulationsmust be prepared in dry conditions, typically at a relative humidity of30% or less. All constituents of the formulation should be predried.

The present invention further provides a pharmaceutical formulation asdefined above for use as an active therapeutic substance, particularlyin the treatment of infection of humans or animals by intracellularpathogens, such as microorganisms of the genus Legionella.

Further the invention provides a method for the treatment of aninfection by intracellular pathogens, such as microorganisms of thegenus Legionella in humans or animals, which comprises administeringthereto, simultaneously or successively in any order, a pharmaceuticallyacceptable salt of clavulanic acid and erythromycin or a derivativethereof, and optionally one or more other antimicrobial agents.Typically the clavulanic acid and erythromycin may be co-administeredtogether, e.g as a composition, optionally also together with one ormore other antimicrobial agents.

Although this invention is not limited to any particular mode ofoperation, there appears to be an in-vivo synergistic interactionbetween the clavulanic acid and the erythromycin against Legionella, andthe combination appears to be capable of penetrating mammalian cells.

Typical microorganisms of the genus Legionella are L. pneumophila. L.micdadei and L. bozemanii are also important pathogens.

The most pharmaceutically stable salt of clavulanic acid is thepotassium salt, ie potassium clavulanate.

Suitable derivatives of erythromycin include the ethylsuccinate,acistrate, estolate, glucoheptonate, propionate, stearate and thelactobionate.

Suitable optional other antimicrobial agents which may be included inthe formulations and methods of this invention as described hereininclude antibiotics, e.g β-lactam antibiotics such as penicillins andcephalosporins. Suitable antibiotics include those for example listed inGB 1578739, e.g on page 3 line 25 to 36 thereof. Preferred β-lactamantibiotics are amoxycillin and ticarcillin, especially amoxycillin,used as the free acid or as a pharmaceutically acceptable salt or ester.Amoxycillin may suitably be used as its trihydrate or sodium salt.Ticarcillin may suitably be used as its sodium salt.

The clavulanic acid and erythromycin and other optional antimicrobialagents, as used in this invention, whether in the form of the freeacids, salts, esters or derivatives thereof are preferably each in asubstantially pure form, e.g. at least 60% pure, more suitably at least75% pure, preferably at least 85% especially at least 98% pure on aweight basis.

The formulations of the invention may be in a form adapted for oral orparenteral use and may be used for the treatment of infection in humansand animals especially mammals, including in particular domesticatedanimals (including farm animals).

The formulations of the invention may, for example, be made up in theform of tablets, suspensions, solutions, reconstitutable powders, andsterile forms suitable for injection or infusion. Such formulations maycontain conventional pharmaceutically acceptable materials, for examplesolid or liquid diluents, colours and preservatives, in accordance withconventional pharmaceutical practice in a manner well understood bythose skilled in the art of formulating antibiotics. Normally all suchingredients are predried. Aqueous solutions are normally provided in theform of dry ingredients for reconstitution immediately prior to use.

It can be particularly advantageous for the formulations of theinvention to be administered to a patient by injection or infusion. Thatmethod of administration has the advantage of rapidly resulting in highblood levels of the active ingredient compounds being administered.Accordingly, in one preferred form of the formulation of the invention,the compounds are present in sterile form, including in sterilecrystalline form. A further preferred form of the formulation of theinvention, is one in which the formulation is in injectable or infusableform.

One injectable or infusable form of the formulation of the invention isan injectable or infusable solution, which suitably comprises an aqueoussolution of a pharmaceutically acceptable salt of clavulanic acid anderythromycin or a derivative thereof, and optionally one or more otherantimicrobial agents, in a sterile pyrogen-free liquid, for examplewater or aqueous ethanol. Because of the water sensitivity of salts ofclavulanic acid such a formulation must be provided as the dryconstituents and be made up with water immediately prior to use.

A further injectable or infusable form of the formulation of theinvention is an injectable or infusable suspension, in which case thesalt of clavulanic acid, and erythromycin or a derivative thereof areadvantageously present in finely particulate form. The suspension may bean aqueous suspension in, for example, sterile water or sterile saline,which may additionally include a suspending agent, for examplepolyvinylpyrrolidone. Alternatively, the suspension may be an oilysuspension in a pharmaceutically acceptable oil suspending agent, forexample arachis oil, which should be dry. Because of the watersensitivity of clavulanic acid salts such aqueous suspensions must bemade up from dry constituents immediately prior to use.

A formulation according to the invention may be in unit dosage form, forexample unit dosage form for parenteral administration, which willprimarily include administration by injection or infusion, especiallyintramuscular and intravenous administration.

In the formulations and methods according to the invention, theerythromycin or derivative thereof may be administered to the patient inan antibacterially effective amount, and the salt of clavulanic acid maybe administered in an amount effective to inhibit β-lactamase enzymes.

The salt of clavulanic acid will generally be administered in an amountsufficient to inhibit the β-lactamase enzyme(s) associated withinfecting bacterial organism(s). To that end, it may suitably beadministered to the patient at a daily dosage of from 0.3 to 15 mg/kg,preferably from 0.7 to 10 mg/kg, for example from 0.7 to 7 mg/kg, ofbody weight. For an adult human (of approximately 70 kg body weight),from 25 to 1000 mg, preferably from 50 to 500 mg, of the salt ofclavulanic acid be administered daily, suitably in from 1 to 6,preferably from 2 to 4, separate doses. Higher or lower dosages may,however, be used in accordance with clinical practice.

When the formulations according to the invention are presented in unitdosage form, each unit dose may suitably comprise from 12.5 to 1000 mg,preferably from 12.5 to 250 mg, of the salt of clavulanic acid. Eachunit dose may, for example, be 12.5, 25, 50, 75, 100, 125, 150, 200, or250 mg of the salt of clavulanic acid.

The ratio of the amount of the salt of clavulanic acid used according tothe invention: amount of any other optional antimicrobial agent presentmay vary within a wide range, e.g 1:1 to 1:30 by weight. In the case ofamoxycillin or salts or esters thereof the said ratio may, for example,be from 1:1 to 1:12; more particularly, it may, for example, be from 1:1to 1:7, 1:1 to 1:4 or 1:1 to 1:2, by weight.

The amount of any optional other antimicrobial agent, e.g amoxycillin orsalts or esters thereof in a formulation according to the invention willnormally be approximately similar to the amount in which it isconventionally used per se In the case of amoxycillin for example from125 to 3000 mg per day, and from 125 to 3000 mg per unit dose,advantageously from about 125 to 1000 mg per unit dose, from 2 to 4times daily may be administered. In the case of ticarcillin for examplea maximum of 3.2 g six to eight hourly may be administered.

The amount of erythromycin or derivative thereof in a formulationaccording to the invention will normally be approximately similar to theamount in which it is conventionally used per se, for example up to 4000mg per day, typically for Legionella treatment 1000-4000 mg per day, forfrom 125-1000 mg per unit dose, administered from 2 to 4 times daily.

An example of a suitable formulation for oral administration accordingto the invention is one comprising from 12.5 to 250 mg, preferably from25 to 125 mg, of potassium clavulanate, in admixture or conjunction with250-4000 mg of erythromycin or a derivative thereof per unit dose,optionally also comprising 125 to 3000 mg of amoxycillin trihydrate.

An example of a suitable formulation for parenteral administrationaccording to this invention is one comprising from 12.5 to 250 mg,preferably from 25 to 125 mg, of potassium clavulanate, in admixture orconjunction with 250-4000 mg of erythromycin or a derivative thereof perunit dose, optionally also comprising 125 to 3000 mg of sodiumamoxycillin.

The following examples illustrate the synergistic antibacterial activityof a salt of clavulanic acid and erythromycin in combination and compareit with the activity of amoxycillin and a salt of clavulanic acid alone,and of erythromycin used alone against Legionella pneumophila.

FIGS. 1 to 5 show graphically the level of L. pneumophila growth invitro following administration of various formulations of the inventioncompared with comparisons and controls.

FIG. 6 shows graphically the level of L.pneumophila growth in vivofollowing administration of formulations of the invention compared withcomparisons and controls.

EXAMPLE 1

Methods:

Human foetal lung fibroblast (MRC-5) cells were growth to 80% confluencyin 6-well plates, using tissue culture medium (TCM). TCM was MinimalEssential Medium with Earles' salts, supplemented with 10% foetal calfserum, 2 mM L-glutamine and 1% non-essential amino acids. The medium wasremoved and replaced with fresh TCM containing 1.0×10⁷ cfu/ml L.pneumophila 1624. The plates were re-incubated for a further 16 h toallow the organisms to become intraceullar.

The inoculant was withdrawn and the infected cells washed to remove anyadherent extracellular bacteria. Fresh TCM containing antibiotics, ordrug-free TCM, was added. Doses were repeated in this way 12, 24 and 36h later.

Amoxycillin trihydrate and lithium clavulanate* were laboratoryreference preparations, and erythromycin lactobionate was a commercialpreparation for injection (Erythrocin, Abbott). The concentrationstested were as follows (μg/ml): amoxycillin (AMX) 4; erythromycin (ERY)2, 0.5, 0.12; amoxycillin/lithium clavulanate acid (AMX/CA) 4/2, 4/1,4/0.5; AMX/CA+ERY 4/1+0.5, 4/1+0.12, 4/05+0.5, 4/0.5+0.12.

At intervals up to 72 h after addition of antibiotics, the infectedcells were washed and lysed with distilled water. Vigorously trituratedlysates were serially diluted in Mueller-Hinton broth and plated ontoBuffered Charcoal Yeast Extract agar (with Legionella growth supplement)and onto 5% blood agar. Colony forming units were enumerated after 3days' incubation.

Results AMX/CA (4/2) was more bactericidal than ERY (2), reducing thenumbers of intracellular legionellae to 2.83×10² cfu/ml by 72 h,compared with 6.67×10³ cfu/ml for ERY (FIG. 1). AMX/CA (4/1) and ERY(0.5) demonstrated equal activity but this was reduced in comparisonwith the higher concentrations (FIG. 2). At 4/0.5 μg/ml and 0.12 μg/mlrespectively, AMX/CA and ERY were bacteristatic only, intracellularorganisms remaining at the level of the starting inoculum for theduration of the test (FIG. 5). A synergistic effect was observed betweenAMX/CA and ERY in combination. AMX/CA (4/1)+ERY (0.5) or (0.12) reducedthe numbers of L. pneumophila to 5.5×10² and 1.37×10³ cfu/ml by 72 h(FIG. 2 & 3), and counts of 2.17×10³ and 8.17×10³ cfu/ml were recoveredfrom cells exposed to AMX/CA (4/0.5)+ERY (0.5) or (0.12) (FIG. 4 & 5).Thus, concentrations of AMX/CA and ERY which were sub-effective alonewere bactericidal in combination, showing activity similar to AMZX/CA(4/2) and ERY (2).

EXAMPLE 2

Materials and Methods 0p Animals: Weanling male rats (60-80 g, CDstrain) were supplied by Charles River UK Ltd.

Induction of leukopenia: Rats were dosed intraperitoneally with 0.5 mlcyclophosphamide (Endoxana, Boehgringer Ingelheim Ltd, Bracknell). at 50mg/kg three days before, and on the day of infection.

Organism: L. pneumophila 1624 (serogroup 1) was used.

Inoculum: L. pneumophila 1624 was grown on BCYEα agar for three days at37° C. and the growth was suspended in Mueller Hinton (MH) broth (BBL).The suspension was standardized using a nephelometer to yield a count of7-8 log 10 cfu/ml, and this was further diluted 1:1000 in MH broth.

Anaesthesia: Rats were anaesthetised by separate intramuscularinjections of 50 μl of fentanyl fluanisone at 0.1 ml/kg(Hypnorm, JanssenPharmaceuticals Ltd., Grove) and diazepam at 0.5 mg/kg(Valium, Rocheproducts Ltd, Welwyn Garden City). The drugs were prepared in steriledistilled water.

Infection: Anaesthetised rats were infected by intrabronchialinstillation of a 50 μl inoculum containing 4-5 log 10 cfu L.pneumophilaby means of non-surgical intratracheal intubation.

Compounds: Amoxycillin trihydrate and potassium clavulanate (SmithKlineBeecham Pharmaceuticals, Worthing) were dissolved in pH8.0 phosphatebuffer and sterile distilled water respectively. Erythromycin base wasdissolved in 10% ethanol and 1% hydroxypropylmethylcellulose (HPMC).

Dosage: Groups of 5 rats received 0.5ml of each agent by oral gavage.Therapy commenced 6 h post infection, and continued q.i.d. for four days(erythromycin t.i.d. because of its longer half life). Rats receivedamoxycillin alone at 200 mg/kg, amoxycillin/potassium clavulanate at200/100 mg/kg, erythromycin at 100 mg/kg, or amoxycillin/potassiumclavulanate acid+erythromycin at 200/100 mg/kg+100 mg/kg.

Results

The study shows a typical L. pneumophila pneumonia in rats (FIG. 6). Theorganism grew well in the rats' lungs, with 5.6±0.9 log 10 cfu/lungsdetectable by 48 h, and 5.9±1.1 log10 cfu/lungs present at 96 h postinfection, although there were no mortalities. Amoxycillin at 200 mg/kgwas ineffective, with numbers of legionellae recovered being similar tothe non-treated control groups throughout the study.Amoxycillin/potassium clavulanate at 200/100 mg/kg and erythromycin at100 mg/kg were equally effective in significantly reducing numbers ofthe organism (p<0.05), with 3.9+1.0 and 3.5±0.8 log 10 cfu/lungsrespectively detectable at 48 h, and 3.9+0.7 and 4.0+1.8 log, 10cfu/lungs at 96 h post infection. However, the combination ofamoxycillin/potassium clavulanate with erythromycin (200/100 mg/kg+100mg/kg) was significantly more effective than either agent alone(p<0.05), in reducing numbers of the organism to 2.4+1.4 log 10cfu/lungs by 24 h, and eliminating the organism to below the limit ofdetection (1.2 log 10 cfu/lungs) by 48 h, with still nothing detectableat 96 h post infection.

I claim:
 1. A pharmaceutical formulation comprising a pharmaceuticallyacceptable salt of clavulanic acid in an amount effective to inhibitβ-lactamase enzymes, and an antibacterially effective amount ofamoxycillin, present as the free acid, or as a pharmaceuticallyacceptable salt or ester thereof and in combination with asynergistically and an antibacterially effective amount of erythromycinor a derivative thereof selected from the group consisting of; theethylsuccinate derivative, the acistrate derivative, the estolatederivative, the glucoheptonate derivative, the propionate derivative,the stearate derivative and the lactobionate derivative.
 2. A method forthe preparation of a pharmaceutical formulation as defined in claim 1which method comprises admixing a pharmaceutically acceptable salt ofclavulanic acid, erythromycin or a derivative thereof selected from thegroup consisting of; the ethylsuccinate derivative, the acistratederivative, the estolate derivative, the glucoheptonate derivative, thepropionate derivative, the stearate derivative and the lactobionatederivative; and amoxycillin.
 3. A method for the treatment of aninfection by a microorganism of the genus Legionella, Chlamydia orMycobacterium in humans or animals, which comprises administeringthereto, simultaneously or successively in any order, a pharmaceuticallyacceptable salt of clavulanic acid in an amount effective to inhibitβ-lactamase enzymes, and an antibacterially effective amount oferythromycin or a derivative thereof selected from the group consistingof; the ethylsuccinate derivative, the acistrate derivative, theestolate derivative, the glucoheptonate derivative, the propionatederivative, the stearate derivative and the lactobionate derivative; andan antibacterially effective amount of an antimicrobial agentamoxycillin, present as the free acid, or as a pharmaceuticallyacceptable salt or ester thereof, provided that the three compounds arepresent at the same time in the body in synergistic amounts.
 4. Acomposition according to claim 1 wherein the pharmaceutically acceptablesalt of clavulanic acid is potassium clavulanate.
 5. A compositionaccording to claim 1 wherein the ratio pharmaceutically acceptable saltof clavalanic acid: amoxycillin is in the range 1:1 to 1:30.
 6. Acomposition according to claim 1 in unit dosage form comprising 12.5 to1000 mg of a pharmaceutically acceptable salt of clavulanic acid and 125to 1000 mg of ertyromycin or a derivative thereof.
 7. A compositionaccording to claim 6 comprising 125 to 3000 mg of amoxycillin or aderivative thereof.